An extreme thermophile Thermus thermophilus is a model organism for structural biology and systems biology, and the so-called “Structural and Functional Whole-Cell Project for T. thermophilus HB8” is in progress. Its genomic sequence composed of a chromosome, a megaplasmid pTT27, and a plasmid pTT8 is available. We revealed that this model organism was a polyploid bacterium harboring four to five copies of the chromosome in a cell. The finding is not surprising, as Deinococcus radiodurans, an extremely radioresistant bacterium closely related to Thermus, is well known to be polyploid. Although the polyploidy of T. thermophilus might allow for genomic DNA protection, maintenance, and repair at elevated growth temperatures, it often complicates the recognition of an essential gene in genetic analyses. We also demonstrated a third plasmid pVV8 in the HB8, and determined its sequence. Limited information and an unfortunate dropout in the substrain, whose genomic sequence was determined, would have prevented the plasmid from coming to public attention. The intrinsic circular plasmid, which was estimated to be six to ten copies in a cell, is 81,151 bp and its G+C content is 68%. The pVV8 was suggested to be nonessential for cell growth.

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The larvae of the sleeping chironomid Polypedilum vanderplanki show an extreme desiccation tolerance, anhydrobiosis, which enable them to survive in an almost completely desiccated state. Upon desiccation, they accumulate a large amount of trehalose and stress-related proteins, such as LEA proteins. LEA proteins would have various roles in cellular protection during drying. Thus, LEA proteins probably contribute to the extreme desiccation tolerance in the larvae. To date, three cDNAs encoding group 3 LEA protein (G3LEA) were cloned from P. vanderplanki. One of the structural characteristics of G3LEA is 11-mer motif repeats. To understand roles of the 11-mer motif, we investigated whether typical features of G3LEA were able to be substituted by a synthesized 22-mer peptide (PvLEA-22) consisting of 2 tandem repeats of the consensus motif of the G3LEAs. Second structure of PvLEA-22 reversibly transformed from random coils in the aqueous solutions into α-helices in the dry state. Such structural transition is similar to that in G3LEA. In addition, PvLEA-22 reinforced glassy matrix of disaccharides, as well as G3LEA. These results indicate that the 11-mer motif structurally governs pivotal roles of G3LEA. PvLEA-22 is expected to become a useful tool for dry preservation of biomaterials and cells at room temperature.

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Bioprocesses under elevated temperatures have several advantages such as reducing risk from contamination, low energy expenditure in agitation and cooling, and facilitating recovery and removal of volatile products. We have focused on a thermophilic bacillus, Geobacillus kaustophilus HTA426, as a potential host for various bioprocesses elevated temperatures and established its genetic modification system. This thermophile is able to grow between 42ºC and 74ºC (optimum, 60ºC) and has some advantages particularly in industrial applications. This review deals with genetic modifications now available in G. kaustophilus HTA426, along with a perspective on thermophiles which are able to be genetically modified and a rational strategy to facilitate the establishment of genetic modification system for desired microbes.

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Acid-extracted cellular polyamines from some newly validated bacterial thermophiles were analyzed by HPLC, GC and GC-MS. A quaternary branched penta-amine, N4-bis(aminopropyl)spermidine, was found in six Thermoanaerobacter species, grown at 60-65℃. A novel tertiary branched penta-amine, N4- aminopropylspermine and a linear penta-amine, thermopentamine, were detected in T. italicus. In four Caldicellulosiruptorspecies, grown at 70℃, N4-bis(aminopropyl)spermidine, N4-bis(aminopropyl)norspermidine and thermopentamine were ubiquitously distributed and caldopentamine, homocaldopentamine and N4-aminopropylspermine were sporadically found. N4-bis(aminopropyl)spermidine spread in Fervidicola, Thermovorax and Ammonifexspecies grown at 70℃ but not in Tepidanaerobacter species grown at 37-50℃, belonging to the order Thermoanaerobacterales. Terrestrial Thermaerobacter compost grown at 70℃ contained caldopentamie and caldohexamine, whereas marine Thermaerobacter species, T. litoralis, T. marianensis and T. nagasakiensis, grown at 70℃, contained thermopentamine, homocaldopentamine, thermohexamine and homocaldohexamine, suggesting that their long polyamine profiles depend on a salt environment as well as growth temperature. Penta-amines and hexa-amines were not found in Thermotalea, Tepidimicrobium, Fervidicella, Caldinitratiruptor and Caloramator species belonging to the order Clostridiales, grown at 50-70℃.

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Shewanella livingstonensis Ac10, a psychrotrophic bacterium isolated from Antarctic seawater, grows well at a temperature range of 4-25˚C. Its major outer membrane protein, named Omp74, is inducibly produced at low temperatures and supposed to play a role in cold adaptation of this bacterium. Omp74 is a homolog of Escherichia coli OmpA, which plays a structural role and forms a channel for hydrophilic solutes in the outer membrane. OmpA has been extensively used as a model to study the folding process of membrane proteins. To characterize Omp74 and analyze its folding process in vitro, it is required to establish in vitro refolding conditions of this protein. In this study, we established the method for purification and refolding of Omp74. Recombinant Omp74 without the signal peptide was overproduced in E. coli, purified, and dissolved with a buffer containing 1% SDS. Then the solution was diluted 50-fold with a buffer containing a detergent. SDS-polyacrylamide gel electrophoresis, tryptophan fluorescence, circular dichroism, and limited proteolysis analyses revealed that refolding was achieved by using a buffer containing various detergents such as 1% octyl β-D-glucopyranoside. We refined the conditions and succeeded in complete refolding of recombinant Omp74 in vitro.

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